MMT, Npeoc-protected spermine, a valuable synthon for the solid phase synthesis of oligonucleotide oligospermine conjugates via guanidine linkers

Article abstract of DOI:10.1016/j.bmc.2011.01.061

Solid phase spermine oligomerization via guanidine linkers was achieved using activated thiourea coupling reaction with primary amino group. Disymmetric spermine synthon was efficiently synthesised in eight steps from spermine. MMT group was used as coupl

Full text of DOI:10.1016/j.bmc.2011.01.061

Bioorganic & Medicinal Chemistry 19 (2011) 1972–1977
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
MMT, Npeoc-protected spermine, a valuable synthon for the solid phase
synthesis of oligonucleotide oligospermine conjugates via guanidine linkers
⇑
Phanélie Perche, Mitsuharu Kotera , Jean-Serge Remy
Laboratoire de Chimie Génétique, UMR7199 CAMB, CNRS et Université de Strasbourg, Faculté de Pharmacie, 67401 Illkirch, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Solid phase spermine oligomerization via guanidine linkers was achieved using activated thiourea cou-
pling reaction with primary amino group. Disymmetric spermine synthon was efficiently synthesised
in eight steps from spermine. MMT group was used as coupling monitor and resulting oligomeric sper-
mines were conjugated to oligonucleotides.
Received 15 October 2010
Revised 26 January 2011
Accepted 28 January 2011
Available online 2 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Guanidine
Oligonucleotide conjugates
Spermine oligomer
Solid phase synthesis
1. Introduction
between spermine units. Guanidine groups are also known to form
high affinity interaction with phosphate groups, thus the resulting
Synthetic vectors based on electrostatic interactions with
nucleic acids and cell membranes such as cationic lipids, cationic
polymers or cationic peptides particles have been extensively used
to carry genetic material into cells. Though efficient in vitro, they
suffer from in vivo limitations for example, aggregation, poor tis-
sue distribution. With the emergence of oligonucleotides as new
tools to control gene expression (antisense oligonucleotides,
siRNA), vectorization can now be performed by oligonucleotides
covalent conjugation to residues responsible for their internaliza-
tion (CPP oligonucleotide conjugates, lipidic oligonucleotides).
Cellular uptake at the single molecule scale instead of particle level
should enhance in vivo distribution.
conjugates may have increased duplex stability. Several efficient
solid phase syntheses of guanidinium oligomer have been de-
scribed.6–11 We thus prepared a new spermine synthon (i.e., 10
in Scheme 1) bearing a MMT group on one NH2 end and a carbam-
oyl protected thiourea group at the distal end. Oligonucleotide–oli-
gospermine conjugates with up to three spermine units were
synthesised by solid phase chemistry. These conjugates were
shown to gain increased duplex stability depending on the number
of spermine introduced.
2. Results and discussion
In our effort to switch from particles to molecules for cell deliv-
ery, we have developed new cationic oligonucleotide-polyamines
conjugates (ZNA i.e., structure 13 in Scheme 2).1,2 These com-
pounds were obtained by repetitive coupling of a natural poly-
amine, the spermine through the well-known phosphoramidite
chemistry. These conjugates exhibited cell penetration potency
and submicromolar activity.3 Fine-tuning the number of coupled
spermines could also provide highly sensitive primers for PCR.4,5
In this article, we present our newly developed solid phase synthe-
sis of oligonucleotide–oligospermine conjugates based on guanid-
inium link formation between spermine units. Such chemistry
allows a faster increase of (cationic groups)/(phosphates) ratio
(N/P ratio) compared to ZNA chemistry containing phosphate links
2.1. Spermine synthon synthesis
The spermine synthon 10 was prepared on a 50 mmol scale
(Scheme 1). Orthogonal protection of primary and secondary
amines was first achieved (to afford intermediate 3). Spermine
was fully trifluoroacetylated to 1 and then treated with Boc anhy-
dride to yield compound 2. The two terminal TFA groups were
spontaneously deprotected by methanol treatment during work-
up to yield 3 (84% three steps-overall yield from spermine). At this
stage, it was shown that TFA groups were not suitable for second-
ary amino group protection. Indeed, Boc deprotection of 3 gave ra-
pid TFA group transposition to afford compound 4.12 Thus, the TFA
groups were replaced by carbamate groups that are not transpos-
able. (Nitrophenyl)ethoxycarbonyl (Npeoc) protecting groups had
been reported to be compatible with oligonucleotide synthesis.13
It was found that the Npeoc group can be readily introduced on
⇑
Corresponding author. Tel.: +33 (0)3 6885 4175; fax: +33 (0)3 6885 4306.
E-mail address: kotera@bioorga.u-strasbg.fr (M. Kotera).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.061

P. Perche et al. / Bioorg. Med. Chem. 19 (2011) 1972–1977
1973
Scheme 1. Spermine synthon synthesis.
pyridine activation. TFA removal of compound 3 by NaOH in a
methanol/water mixture afforded 5 and introduction of Npeoc
group was then achieved by the carbonate 6 with DMAP to provide
7. These steps were optimised to give excellent yields (>91% pro
step) without using tedious column chromatography and the prod-
uct 7 was shown to be of good purity, as revealed by elementary
analysis. Boc groups of the primary amines of 7 were subsequently
deprotected by TFA treatment (12 equiv) and crude TFA salts were
treated by 4-monomethoxytrityl chloride (MMTCl; 4.2 equiv), with
excess of triethylamine (20 equiv) to afford bis-MMT product 8 in
good yield (79%). Partial deprotection of MMT gave the desired
mono MMT amine 9 and unreacted bis-MMT product 8 was recov-
ered and recycled to afford overall yield up to 62% after three cy-
cles.14 Finally the reactive thiourea was inserted by addition of 9
to a solution of Fmoc isothiocyanate in dichloromethane at 0 °C
to afford 10 (80%) after flash column chromatography purification,
as a white, easy-to-handle foam.
2.2. Oligonucleotide–oligospermine conjugates synthesis
Spermines were oligomerized on solid support prior to oligonu-
cleotide synthesis (Scheme 2).15 dT-CPG was first conjugated to a
diaminolinker (C8) by successive treatment of TCA, carbodiimide
and then mono MMT-protected diaminooctane 11. According to
trityl assay, the conjugation efficiency was 75–80%. In order to stop
further participation of un-reacted sites, the solid support was fi-
nally treated in prolonged capping conditions (Ac2O, NMI, 6 min).
Several reagent-base combinations were examined for couplings
of spermine synthon 10 including DCC, EDC, EDC methiodide and
Mukaiyama reagent with the following bases: DIPEA, pyridine or
NMI. The best results were obtained using 20 equiv synthon 11
with EDCꢀHCl activation (88 equiv) in presence of DIPEA (88 equiv)
for 4 h. The coupling yields determined by trityl assay attained
88%.16 Unreacted sites were equally capped using Ac2O/NMI re-
agent. The same protocol was used for the second and third cou-
pling, affording similar coupling yields. The last MMT group was
then detritylated and on-machine ODN synthesis was performed
in standard conditions, except for the first coupling that used pro-
longed coupling time and repeated amidite delivery.17 The oligo-
nucleotide–spermine conjugates were cleaved from CPG and base
deprotection was carried out by concd NH4OH at room tempera-
ture for 24 h. Ammonia solutions were lyophilized and the residual
crude materials were suspended in DBU 1 M/acetonitrile (55 °C
3 days at rt) for Npeoc deprotection.18 After desalting, crude prod-
Scheme 2. Oligonucleotide–oligospermine conjugates synthesis.
secondary amine by the use of 4-nitrophenethyl 4-nitrophenyl-
carbonate (6). Thus, we prepared the carbonate 6 by reaction of
4-nitrophenyl chloroformate with 4-nitrophenethyl alcohol under

1974
P. Perche et al. / Bioorg. Med. Chem. 19 (2011) 1972–1977
Table 1
standard DNA phosphoramidites, with standard capping reagents
(cap Mix A: Ac2O/THF 1:9, Cap mix B: 10% N-methylimidazole/
THF/pyridine: 1:8:1), 5-benzylthio[1H]tetrazole in acetonitrile as
activator and 0.02 M I2 in THF/pyridine/H2O as oxidizing solution.
Melting temperatures (Tm, °C) of duplexes containing conjugates 12a–12ca
b
ODN
Tm (°C)
DTm (°C)
12a
12b
12c
13a
13b
13c
14
49.3
48.3
50.4
48.1
49.3
53.3
46.4
+2.9
+1.9
+4.0
+1.7
+2.9
+6.9
Ref.
4.1.2. Analyses and purifications
Melting points were measured in a Stuart Scientific SMP2 melt-
ing point apparatus 2 and are uncorrected. IR spectra were per-
formed on a Nicolet 380 FT-IR with a Smart Orbit ATR Sampling
Accessory from ThermoFischer. 1H and 13C spectra were performed
either on a 400 MHz Bruker Avance III instrument (BBFO+ probe), a
200 MHz Bruker DPX spectrometer, or a 300 MHz Bruker Avance
DPX spectrometer with dual (1H, 13C) probe. NMR chemical shifts
are reported as ppm relative to tetramethylsilane and converted
to the TMS scale using the residual proton of the corresponding
deuterated solvent. Data for 1H NMR spectra are reported as fol-
lows: multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
quintuplet, m = multiplet, dt = double triplet, br = broad), coupling
constants, integration. Data for 13C NMR spectra are reported as
follows: DEPT attribution, integration. Due to the C–N bond rota-
tion retardation in trifluoroacetyl or carbamate functions, many
13C signals were split in several peaks. Elementary analyses were
performed by the CNRS Service Central d’Analyses, USR-59 on
homemade microanalyzers. Mass spectrometry was carried out
with positive ionization either on a LC–MS spectrometer Agilent-
1200 (LC) MM-ESI-APCI-MSD (MS), a MS Bruker (Ion Trap, HCT Ul-
tra, ESI) or a MS Agilent (QTof 6520, ESI) spectrometer. Oligonu-
cleotide mass spectra (MALDI-TOF) were obtained in the positive
mode on a Bruker Ultraflex apparatus with hydroxypicolinic acid
combined with diammonium citrate as matrix. UV/vis. absorption
spectra were recorded on a Varian Cary 100 bio spectrophotome-
ter. Merck Silica Gel 60 F254 plates were used for analytical thin
layer chromatography. TLC analysis was facilitated by the use of
the following stains in addition to UV light (254 nm) with fluores-
cent-indicating silica gel plates: 5% phosphomolybdic acid/EtOH or
a
Melting temperatures were determined using a 1.0
lM duplex concentration in
1 mL buffer composed of 10 mM HEPES pH 7.4, 0.15 M NaCl.
b
Tm increase compared to the natural duplex (14).
ucts 12a and 12b were shown to be of good purity without further
purification. For 12c, the final DMT group was kept during ODN
synthesis and C18-based purification was carried after the same
deprotection procedure to afford the final product. The conjugates
12a–c were characterized by MALDI-TOF mass spectrometry.
Melting temperatures of duplexes containing the oligonucleo-
tide conjugates 12a–c were determined and compared with those
containing ZNA 13a–c prepared in our laboratory (Table 1).2,3 The
natural duplex melted at Tm = 46.4 °C. As expected,
DTm of guani-
dine linked spermine conjugates 12a is higher than corresponding
ZNA (+2.9 °C vs +1.7 °C). Conjugates containing two and three sper-
mines units (12b and 12c) show equally increased duplex stability
compared to the natural duplex however stabilizing effect
DTm/
(number of spermine units) for 12b and 12c were lower in these
cases than that of ZNA. Surprisingly the second conjugation of
spermine unit is shown to be destabilizing for duplex
(Tm = 48.3 °C for 12b vs Tm = 49.3 °C for 12a). The reason of this
observation is still not understood. Addition of 3 spermines (12c
and 13c) resulted in
DTm in favor of phosphate linkages (+4.0 vs
+6.9 °C, respectively). We speculate that guanidine linkage being
more rigid than phosphate, the resulting oligocation might not
bend over the duplex to stabilize it.
5% ninhydrin/EtOH. Merck Silica Gel 60 (particle: 40–63 lm) was
employed for column chromatography separations. Oligonucleo-
tide purifications were performed on PolyPak II reverse phase car-
tridges (Glen Research). Oligonucleotides were freeze-dried on a
Savant Speed Vac concentrator SVC 100H from Thermo Scientific.
3. Conclusion
We developed efficient synthesis of spermine synthon 10. We
successfully oligomerized this synthon 10 on CPG support and
the resulting oligospermine was successfully conjugated to oligo-
nucleotide. Increased duplex stability was observed for these con-
jugates. Current study addresses the improvement of coupling
efficiency of guanidine linkage and deprotection procedures in or-
der to allow increased number of spermine couplings.
4.2. Synthesis of spermine synthon
4.2.1. N1,N2,N3,N4-Tetra(trifluoroacetyl)spermine (1)
Spermine (10 g, 50 mmol) and DMAP (37.2 g, 300 mmol) were
stirred in DCM (200 mL) under argon and cooled to 0 °C. Trifluoro-
acetic anhydride (52.3 g, 250 mmol) in DCM (150 mL) was added
dropwise over 1.5 h. The ice bath was then removed and the mix-
ture was allowed to stir at rt for 5.5 h. The reaction was quenched
by the addition of dist. H2O (350 mL). The aqueous layer was sep-
arated and extracted with DCM (2 ꢁ 200 mL). Organic phases were
combined, dried for 15 min (MgSO4), filtered and concentrated in
vacuo to give a colourless oil (39.4 g) containing residual DMAP.
The crude product was used without further purification for the
synthesis of 3. The pure product could be obtained by salinification
for analyses: the oil was dissolved in ethanol by heating with a
heat gun. HCl (37%) was added and the mixture was cooled to
0 °C. The product was filtered, washed with dist. H2O and dried
overnight in vacuo before analyses. Analyses were performed on
the saline product.
4. Experimental section
4.1. General
4.1.1. Reagents and solvents
Unless otherwise stated, all reagents were purchased from Sig-
ma Aldrich, TCI and Merck and used without further purification.
All solvents were purchased from Carlo Erba reagents-SDS or
VWR-Prolabo. All synthesized compounds were characterized
using standard analytical and spectroscopic techniques. Oligonu-
cleotides were prepared on an automated Expedite 8900 Nucleic
Acid Synthesis System (GMI Inc, USA) at 1
l
mol scale. 30-dT-CPG
1
l
mol columns, (50-dimethoxytrityl-30-thymidine, 20-succinoyl-
White powder, mp: 114 1 °C; IR: = 1734.5
m
= 3317.8 (N–H),
m
long chain alkylamino-CPG) were purchased from Glen Research.
Oligonucleotide synthesis reagents, solvents and purification car-
tridges were all purchased from Glen Research/Eurogentec (Paris,
France) except for external acetonitrile (Wash A), which was pro-
vided by Carlo Erba reagents-SDS. All phosphoramidites used were
(C@O), ;
m
= 1677.8 (C@O), d = 1561.4 (N–H) cmꢂ1 1H NMR
(400 MHz, DMSO) d 9.51 and 9.41 (2t, J = 5.6 Hz, 2H), 3.40–3.36
(br m, 8H), 3.25–3.18 (m, 4H), 1.86–1.72 (m, 4H), 1.63–1.43 (br
m, 4H); 13C NMR (400 MHz, DMSO) d 156.13–156.01, 155.76–
155.65 (2C), 155.21, 154.86 (2C), 120.16–119.77, 117.30–116.88,

P. Perche et al. / Bioorg. Med. Chem. 19 (2011) 1972–1977
1975
114.43–114.02, 111.57–111.17 (4C), 46.81–46.70, 46.06–45.88,
44.69–44.64, 44.24 (CH2, 4C) 36.87, 36.45 (CH2, 2C), 27.74, 25.83
(CH2, 2C), 25.43–25.15, 23.56–23.34 (CH2, 2C); LC–MS, m/z: calcd
for C18H22F12N4O4: 586.1, found: 587.0 [M+H]+.
(quintuplet, J = 8.8 Hz, 4H), 1.52–1.48 (br m, 4H), 1.41 (s, 18H).
13C NMR (400 MHz, CDCl3) d 156.21 (2C), 78.90 (2C), 49.78 (2C),
47.72 (2C), 39.24 (2C), 29.97 (2C), 28.52 (6C), 27.87 (2C); Anal.
Calcd for C20H42N4O4: C, 59.67; H, 10.52; N, 13.92. Found: C,
59.53; H, 10.44; N, 13.76. LC–MS m/z: calcd for C20H42N4O4:
402.3, found: 403.3 [M+H]+.
4.2.2. N1,N4-Bis(tert-butoxycarbonyl)-N2,N3-bis(trifluoroacetyl)
spermine (3)
Di(tert-butyl) dicarbonate (33.7 g, 150 mmol) in DCM (100 mL)
was added dropwise to a suspension of the crude product 1 (39.4 g)
and DMAP (12.4 g, 100 mmol) in DCM (180 mL). During the reac-
tion, a gas release was observed and the suspension solubilised.
The reaction was stirred overnight at rt. 1 M aqueous NH4Cl
(200 mL) was added; the organic layer was separated and washed
with 1 M NH4Cl (2 ꢁ 200 mL). The organic phase was then dried for
15 min (MgSO4), filtered off and concentrated under reduced pres-
sure. The crude product was recrystallised under stirring in meth-
anol (60 mL), cooled to 4 °C for 1 h, filtered off, washed with cold
methanol and dried in vacuo overnight. White powder: 24.4 g,
4.2.5. 4-Nitrophenethyl 4-nitrophenyl carbonate (6)
4-Nitrophenyl chloroformate (21.8 g, 105 mmol) in DCM
(140 mL) was cooled to 0 °C under stirring. A mixture of 4-nitro-
phenethyl alcohol (16.9 g, 100 mmol) and pyridine (9 mL,
111 mmol) in DCM (60 mL) was added dropwise at 0 °C within 2 h.
The reaction was then allowed to warm to rt until consumption of
allthe4-nitrophenethylalcoholasrevealedbyTLC(AcOEt/cyclohex-
ane 1:1). The reaction mixture was washed by 1 M Na2CO3 (200 mL)
and by dist. H2O (2 ꢁ 200 mL). The organic layer was then dried for
15 min (MgSO4), filtered and evaporated under reduced pressure.
Precipitation with diethyl ether (300 mL) and subsequent cold
diethyl ether washes yielded 6. Off-white powder: 25.4 g, 76.5%,
84% (steps 1, 2 and 3), mp: 141 1 °C; IR:
m
= 3344.8 (N–H),
m
= 1693.4 (C@O),
m
= 1675.5 (C@O), d = 1519.9 (N–H) cmꢂ1
;
1H
mp: 104 1 °C; IR:
= 1518.7 (NO2 as),
m
m
= 3084.9 (Ar C–H),
m
= 1766.3 (C@O),
NMR (400 MHz, CDCl3) d 5.01 (br, 1H), 4.79 (br, 1H), 3.40–3.34
(br m, 8H), 3.12–3.05 (m, 4H), 1.79–1.68 (m, 4H), 1.56–1.54 (br
m, 4H), 1.381 (s, 18H); 13C NMR (400 MHz, CDCl3) d 157.39–
157.33, 157.2–157.17, 157.03–156.98, 156.85–156.82 (2C),
156.24 (2C), 120.89, 118.05, 115.19, 112.31 (2C), 79.70–79.60,
79.42–79.34 (2C), 47.15, 46.43–46.36, 45.49, 44.43–44.38 (4C),
37.91–37.67 (2C), 29.90–29.76, 27.65–27.58 (2C), 28.51–28.45
(6C), 25.99–25.92, 24.15–24.06 (2C); Anal. Calcd for C24H40F6N4O6:
C, 48.48; H, 6.78; N, 9.42. Found: C, 48.35; H, 6.87; N, 9.25. LC–MS,
m/z: calcd for C24H40F6N4O6: 594.3, found: 617.2 [M+Na]+.
m
= 1346.7 (NO2 sym) cmꢂ1
;
1H NMR
(400 MHz, CDCl3) d 8.25–8.15 (m, 4H), 7.44–7.29 (m, 4H), 4.54–
4.51 (t, J = 6.8 Hz, 2H), 3.19–3.13 (t, J = 6.6 Hz, 2H); 13C NMR
(200 MHz, CDCl3) d 155.45 (1C), 152.38 (1C), 147.17 (1C), 145.51
(1C), 144.79 (1C), 129.97 (C–H, 2C), 125.38 (C–H, 2C), 123.95 (C–H,
2C), 121.84 (C–H, 2C), 68.63 (CH2, 1C), 34.80 (CH2, 1C); Anal. Calcd
for C15H12N2O7: C, 54.22; H, 3.64; N, 8.43. Found: C, 54.18; H, 3.6;
N, 8.5. HRMS (ESI) m/z: calcd for C15H12N2O7: 332.064, found:
332.064.
4.2.6. N1,N4-Bis(tert-butoxycarbonyl)-N2,N3-bis[2-(4-
nitrophenyl)ethoxycarbonyl]spermine (7)
4.2.3. N1,N4-Bis(trifluoroacetyl)spermine (4)
To a suspension of spermine 3 (596 mg, 1 mmol, 1 equiv) in
solution in DCM (5 mL) were added slowly under stirring 2 mL of
trifluoroacetic acid. Two phases appeared. After 30 min of reaction,
the mixture was evaporated to provide an orange oil (1.31 g). The
product was then gently washed and triturated in DCM (3 ꢁ 2 mL)
and diethyl ether (3 ꢁ 2 mL) to eliminate residual TFA. Organic sol-
vents were then removed with a pasteur pipette. The product was
then dried by evaporation under reduced pressure. To provide 4 as
a free amine, the product (552 mg) dissolved in MeOH (3.5 mL)
was passed through an ion exchange column (Dowex 21KCl ex-
change anion, 3.6 g) that had been previously prepared by succes-
sive elution of an aqueous solution of NaOH 1 M (300 mL), distilled
water (until pH 7) and methanol (50 mL). The product was then
eluted with methanol and evaporated under reduced pressure to
yield 4 as a pale yellow oil (324 mg, 55%). 1H NMR (300 MHz,
CDCl3) d 9.48 (br, 2H), 3.47–3.43 (t, J = 6 Hz, 4H), 2.84–2.80 (t,
J = 5.4 Hz, 4H), 2.61–2.56 (t, J = 6.3 Hz, 4H), 1.72–1.65 (m, 4H),
1.50–1.46 (m, 4H).
To 5 (15.5 g, 39 mmol) in DCM (115 mL) were added at rt, under
stirring6(26.9 g, 81 mmol)andDMAP(4.9 g, 40 mmol). Thereaction
mixture was allowed to stir overnight. 1 M aqueous Na2CO3
(200 mL) was added and salts appeared in the aqueous layer. The
aqueous phase was diluted with dist. H2O (100 mL). The organic
layer was separated, washed with 1 M Na2CO3 (2 ꢁ 200 mL), dried
for 15 min (MgSO4), filtered off and evaporated under reduced pres-
sure. Pure product 7 was obtained by precipitation in diethyl ether
(300 mL), filtered off, washed with cold ether and dried overnight
in vacuo. White powder: 29.4 g, 97%, mp: 134 1 °C; IR:
(N–H), = 1694.1 (C@O), = 1519.2 (NO2 as), = 1345.7 (NO2
sym) cmꢂ1 1H NMR (400 MHz, CDCl3) d 8.11–8.09 (d, J = 8.4 Hz,
m = 3349.7
m
m
m
;
4H), 7.34–7.32 (m, 4H), 5.11 (br, 1H,), 4.60 (br, 1H), 4.30–4.27 (t,
J = 6.6 Hz, 4H), 3.19–2.93 (m, 16H), 1.58–1.53 (m, 4H), 1.37 (s,
18H); 13C NMR (400 MHz, CDCl3) d 156.39–155.85 (4C), 146.93
(2C) 146.15 (2C), 129.85 (4C), 123.76 (4C), 79.47–79.07 (2C), 64.92
(2C), 47.11, 46.53 (2C), 44.52 (2C) 38.13, 37.43 (2C), 35.49 (2C),
29.22 (2C), 28.51 (6C), 25.86–25.22 (2C); Anal. Calcd for
C
38H56N6O12: C, 57.85; H, 7.15; N, 10.65. Found: C, 57.68; H, 7.35;
4.2.4. N1,N4-Bis(tert-butoxycarbonyl)spermine (5)
N, 10.60. HRMS (ESI) m/z: calcd for C38H56N6O12: 788.396, found:
To a suspension of 3 (24.4 g, 41 mmol) in methanol (130 mL)
was added, at rt, a 10 M aqueous solution of NaOH (40 mL). The
reaction was allowed to proceed overnight under stirring. The fol-
lowing day, the mixture was concentrated under reduced pressure
to a third of its initial volume and supplemented with 1 M aqueous
NaOH (65 mL). DCM (150 mL) was added; the aqueous layer was
separated and extracted with DCM (2 ꢁ 150 mL). The organic lay-
ers were combined, dried for 15 min (MgSO4), filtered and concen-
trated under reduced pressure. The pure product was obtained by
precipitation with heptane (200 mL) and dried in vacuo overnight
before analyses. White powder: 15.5 g, 94%, mp: 94 1 °C; IR:
788.396.
4.2.7. N1,N4-Bis(monomethoxytrityl)-N2,N3-bis[2-(4-nitro
phenyl)ethoxycarbonyl]spermine (8)
To a stirred solution of 7 (29.4 g, 37 mmol) in DCM (180 mL)
cooled at 0 °C was slowly added trifluoroacetic acid (44.4 g,
390 mmol) over 5 min. The reaction was next allowed to warm up
to rt within 2 h. The reaction mixture was evaporated under reduced
pressure. The crude deprotected product containing remaining tri-
fluoroacetic acid was then dissolved in DCM (100 mL) and cooled
to 0 °C under stirring. A large excess of triethylamine (72.6 g,
720 mmol) in DCM (50 mL) was added dropwise. The ice bath was
then removed and 4-monomethoxytrityl chloride (48.2 g,
156 mmol) in DCM (100 mL) was rapidly added at rt. After 30 min,
m
= 3344.8 (N–H), m ;
= 1693.1 (C@O), d = 1522.1 (N–H) cmꢂ1 1H
NMR (300 MHz, CDCl3) d 5.16 (br, 2H), 3.20–3.14 (q, J = 8 Hz, 4H),
2.66–2.62 (t, J = 8.8 Hz, 4H), 2.60–2.56 (t, J = 8 Hz, 4H), 1.66–1.57

1976
P. Perche et al. / Bioorg. Med. Chem. 19 (2011) 1972–1977
1 M aqueousNa2CO3 (150 mL)was poured into the reaction mixture.
The aqueous phase was separated and extracted with DCM
(2 ꢁ 150 mL). Combined organic layers were dried for 15 min
(MgSO4), filtered and evaporated to dryness. The crude product (yel-
low oil, 60 g), was purified by flash column chromatography on silica
gel using AcOEt/cyclohexane: 1:1 as eluent to afford 8, dried over-
CDCl3) d 9.89 (br s, 1H), 9.59 (br s, 1H), 8.10–8.04 (m, 4H), 7.77–
7.75 (d, J = 7.6 Hz, 2H), 7.54–7.52 (d, J = 7.6 Hz, 2H), 7.42–7.38 (m,
6H,), 7.33–7.28 (m, 8H), 7.26–7.23 (m, 4H), 7.17–7.14 (br m, 2H),
6.79–6.76 (br m, 2H), 4.47–4.46 (d, J = 6.8 Hz, 2H), 4.34–4.20 (m,
5H), 3.74 (s, 3H), 3.64–3.52 (br m, 2H), 3.26–2.95 (br m, 12H),
2.07–2.04 (br m, 2H), 1.82–1.33 (br m, 8H); 13C NMR (400 MHz,
CDCl3) d 179,24 (1C), 158.01–157.91 (1C), 155.89 (2C), 156.59,
152.81–152.46, 148.96 (1C), 146.93–146.89 (2C), 146.55, 146.37,
146.20, 146.03, (4C), 143.03 (2C), 141.43 (2C), 138.46 (1C), 129.83
(CH, 4C), 128.60 (CH, 6C), 128.23 (CH, 2C), 127.94–127.86 (CH, 4C),
127.39 (CH, 2C), 126.42–126.22 (CH, 2C), 125.01 (CH, 1C), 123.79–
123.77 (CH, 4C), 120.35 (CH, 2C), 113.24 (CH, 2C), 70.51 (1C),
68.49–68.37 (CH2, 1C), 65.03–64.77 (CH2, 2C), 55.31 (CH3, 1C),
46.42, 46.81, 47.14, 46.42 (CH2, 2C), 46.63 (CH, 1C), 45.51 (CH2,
1C), 44.65 (CH2, 1C), 42.81–43.02 (CH2, 1C), 41.33–40.85 (CH2, 1C),
35.50 (CH2, 2C), 30.07, 29.22, 27.48–27.05, 25.87–25.44 (CH2, 4C);
HRMS (ESI) m/z: calcd for C64H67N7O11S: 1141.46, found: 1142.47
[M+H]+.
night in vacuo. White foam (33.5 g, 79%); IR:
m
= 1689.9 (C@O),
m
= 1517.8 (NO2 as), ;
m
= 1344.1 (NO2 sym) cmꢂ1
1H NMR
(400 MHz, CDCl3) d 8.15–8.04 (m, 4H), 7.43–7.41 (d, J = 7.6 Hz, 8H),
7.33–7.32 (d, J = 8.4 Hz, 4H), 7.28–7.23 (m, 12H), 7.18–7.13 (m,
4H), 6.79–6.75 (m, 4H), 4.25–4.22 (br m, 4H), 3.74 (s, 6H), 3.27–
2.94 (br m, 12H), 2.05 (br m, 4H), 1.66–1.30 (br m, 8H); 13C NMR
(400 MHz, CDCl3) d 158.04–157.94 (2C), 155.90 (2C), 146.92 (4C),
146.57, 146.41, 146.22 (4C), 138.48–138.29 (, 2C), 129.85 (4C),
128.63 (12C), 127.97 (8C), 126.45–126.26 (4C), 123.79 (4C), 113.27
(4C), 70.30 (2C), 64.78 (2C), 55.33 (2C), 47.24–46.46 (2C), 45.51
(2C), 41.36–40.87 (2C), 35.54 (2C), 30.08–29.26 (2C), 25.92–25.51
(2C); HRMS (ESI) m/z: calcd for C68H72N6O10: 1132.531, found:
1132.532.
4.2.10. N1-(Monomethoxytrityl)diaminooctane (11)
4.2.8. N1-(Monomethoxytrityl)-N2,N3-bis[2-(4-
nitrophenyl)ethoxycarbonyl]spermine (9)
To a solutionof diaminooctane (7.2 g, 50 mmol)dissolved inDCM
(70 mL) was added dropwise at 0 °C, under stirring, a solution of 4-
monomethoxytrityl chloride (3.3 g, 10 mmol) dissolved in DCM
(200 mL). The reaction was then allowed to warm up to room tem-
perature for 1.25 h. An aqueous solution of Na2CO3 1 M (200 mL)
was added. The aqueous phase was extracted by DCM
(2 ꢁ 100 mL). Organic phases were pooled, dried on MgSO4, filtrated
and evaporated under reduced pressure. The crude product (6.8 g)
was purified by flash chromatography (eluent: DCM/MeOH/Et3N:
90:10:2). The pure product 11 was obtained as a white oil (2.7 g,
65%). 1H NMR (400 MHz, CDCl3) d 7.46–7.43 (m, 4H), 7.37–7.33 (m,
2H), 7.26–7.22 (m, 4H), 7.17–7.12 (m, 2H), 6.80–6.76 (m, 2H), 3.75
(s, 3H), 2.69–2.66 (t, J = 7.1 Hz), 2.11–2.07 (t, J = 7 Hz, 2H), 1.48–
1.40 (m, 4H), 1.30–1.21 (m, 8H); 13C NMR (400 MHz, CDCl3) d
157.93 (1C), 146.81 (2C), 138.75 (1C), 129.97 (2C), 128.75 (4C),
127.86 (4C), 126.22 (2C), 113.19 (2C), 70.53 (1C), 55.34 (1C), 43.75
(1C), 42.03 (1C), 33.04 (1C), 31.06 (1C), 29.76 (1C), 29.55 (1C),
27.50 (1C), 26.98 (1C).
To a stirred solution of 8 (33.5 g, 30 mmol) in DCM (200 mL) was
poured trifluoroacetic acid (6.8 g, 59 mmol) in DCM (200 mL) at rt.
The mixture immediately turned orange which went intensifying
in the course of the reaction. Reaction was monitored by TLC until
the orange color of the mono-trityl protected product spot appeared
nearly a half as intense as the color of the bis-trityl protected product
spot (1 h reaction time, TLC eluent: DCM/MeOH/NH3, 90:10:1, stain-
ing of the plate: 5% PMA/EtOH). 1 M aqueous Na2CO3 (400 mL) was
then added to quench the reaction. The aqueous layer was extracted
by DCM (2 ꢁ 200 mL). The combined organic phase was dried for
15 min (MgSO4), filtered off and evaporated under reduced pressure.
The crude product (yellow oil, 36 g) was purified by flash column
chromatography on silica gel using a 90:10:0–80:20:6 gradient of
DCM/MeOH/NH3 (2 M in methanol), which yielded the pure product
(6.8 g, 8%) as a pale yellow foam. The reaction was reiterated on
unreacted fractions of 8 until the global yield reached 62% of the in-
volved material. In one step, yield of the reaction could be levelled up
to 37% (10 mmol scale, 2.1 equiv of trifluoroacetic acid, 55 min reac-
tion time). Pale yellow foam: 16.0 g, 62%; 1H NMR (400 MHz, CDCl3)
d 8.16–8.07 (dd, J1 = 29.2 Hz, J2 = 8 Hz, 4H), 7.47–7.45 (d, J = 8 Hz,
4H), 7.37–7.25 (m, 10H), 7.20–7.17 (m, 2H), 6.83–6.81 (m, 2H),
4.35–4.29 (dt, J1 = 18.8 Hz, J2 = 6.4 Hz, 4H), 3.77 (s, 3H), 3.31–2.97
(m, 12H), 2.74–2.63(m, 2H), 2.10 (br m, 2H), 1.71 (br m, 2H), 1.60–
1.27 (br m, 8H); 13C NMR (400 MHz, CDCl3) d 157.96–157.85 (1C,
1), 155.85 (2C), 146.87–146.82 (2C), 146.49, 146.32, 146.18 (4C),
138.40–138.19 (1C), 129.83 (4C), 128.54 (6C), 127.88 (4C), 126.35–
126.18 (2C), 123.70 (4C), 113.18 (2C), 70.45 (1C), 64.95–64.75 (2C),
55.24 (1C), 47.17–47.08, 46.53–46.37, 45.44 (6C), 41.27–40.78
(1C), 35.46 (2C), 31.98 (1C), 30.45–29.16 (1C), 25.83–25.38 (2C);
MS (ESI), m/z: calcd for C48H56N6O9: 860.99, found: 861.40, [M+H]+.
4.3. General procedure for the synthesis of spermine oligomers
on solid support
4.3.1. Amino linker introduction on dT-CPG
A 1 lmol scale dT-CPG column was automatically detritylated on
an Expedite synthesizer by a solution of TCA in DCM (Glen Research
reagent). DMT fraction was collected and diluted in 3% TCA/DCM
solution (100 mL) and its absorbance at 503 nm was measured.
The column was then manually rinsed with DCM (3 ꢁ 1 mL) and
flushed under argon. A solution of CDI (32 mg, 200 lmol) in anhy-
drous DCM (1 mL) was percolated trough for 1 h. The reagent was
then flushed out of the system; the cartridge was rinsed with DCM
(2 ꢁ 1 mL) and flushed under argon. A solution of 11 (50 mg,
120 lmol) and triethylamine (12.3 mg, 120 lmol) in anhydrous
4.2.9. N1-(9-Fluorenylmethoxycarbonylthiourea)-N2,N3-bis[2-
(4-nitrophenyl)ethoxycarbonyl]-N4-
(monomethoxytrityl)spermine (10)
To a stirred solution of Fmoc-isothiocyanate (6.4 g, 23 mmol) in
DCM (300 mL) cooled at 0 °C was added 9 (12.7 g, 15 mmol) under
argon. The reaction occurred instantaneously as revealed by TLC
(eluent: AcOEt/cyclohexane, 1:1, staining agent: PMA 5% in EtOH).
The ice bath was then removed and the reaction mixture was evap-
orated under reduced pressure. The crude product was purified by
flash column chromatography on silica gel (eluent: AcOEt/cyclohex-
ane, 1:1), which afforded the pure product 10, dried overnight in va-
DCM (1 mL) were percolated through the column for 2 h and the re-
agents were flushed out of the system. The column was washed with
DCM (3 ꢁ 1 mL) and flushed under argon. Unreacted sites were
capped by Ac2O/NMI on a synthesizer with a prolonged capping cy-
cle of 6 min. The MMT final protecting group was detritylated on the
synthesizer with the following protocol: a TCA/DCM solution (Glen
Research reagent) was passed through the column during 15 s, the
reaction was allowed to proceed for 3 min and the cycle was re-
peated four times (16 min). The column was then rinsed with aceto-
nitrile (two washes) and flushed under argon. MMT fraction was
collected and diluted in 3% TCA/DCM solution (100 mL) and its
absorbance at 478 nm was measured. Yields of amino-linker cou-
pling step were calculated on MMT absorbance at 478 nm and
cuo. White foam: 13.9 g, 82%; IR:
m
m
= 3294.0 (NH),
m = 1692.7 (C@O),
= 1518.0 (NO2 as), 1344.7 (NO2 sym) cmꢂ1
;
1H NMR (400 MHz,

P. Perche et al. / Bioorg. Med. Chem. 19 (2011) 1972–1977
1977
DMT absorbance at 503 nm ratios, corrected by a UV ratio factor
(UVRF) of 1.36 (EDMT = 76 mL cmꢂ1 molꢂ1, EMMT = 56 mL cmꢂ1
molꢂ1).22 Amino-linker coupling yields were comprised between
75% and 80%.
4.6. Melting temperature (Tm) studies
l
l
Melting temperature studies were carried out in 1 cm path
length quartz cells on a CARY 100Bio UV–vis spectrophotometer
(Varian, France) by measuring the absorption at 260 nm. The sam-
ples were placed in a CARY Thermostatable Multicell Holder acces-
sory (Varian) and the temperature was regulated by a CARY
Temperature controller by Peltier effect (Varian). Samples for anal-
4.3.2. Spermines oligomerisation on amino-modified dT-CPG
A 0.4 M solution of DIPEA in DCM (1 mL) was passed through
the column. Before that, 10 (23.5 mg, 20
88 mol) were dried for 30 min in vacuo. 10 and EDC were then
mixed and dissolved in 0.4 M DIPEA (220 L, 88 mol) before being
lmol) and EDC (17 mg,
l
ysis consisted of a 1.0 lM concentration of each oligonucleotide in
l
l
1 mL buffer composed of 10 mM HEPES pH 7.4, 0.15 M sodium
chloride. All samples were annealed by heating at 95 °C for
20 min before slowly cooling down to 20 °C The experiments were
carried out by increasing the temperature at a rate of 0.7 °C per
minute from 20 to 90 °C and the temperature was recorded every
0.5 °C. Tm values were taken as the maximum of the first deriva-
tive absorption curve versus temperature.
percolated through the column for 4 h. The reagents were flushed
out and the column was rinsed by DCM (3 ꢁ 1 mL). The active sites
that had not reacted during the process were capped manually
with 0.5 mL of acetic anhydride (cap Mix A, Glen Research)/
0.5 mL N-methylimidazole (cap mix B, Glen Research reagent) for
30 min. The final MMT protecting group was removed following
the same procedure as for amino-linker couplings. Couplings of
additional spermine residues on the resin were equally performed.
Yields of spermine coupling steps were calculated on MMT absor-
bances ratio at 478 nm and were comprised between 70% and 88%.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.061.
4.4. General procedure for automated oligonucleotide
syntheses on solid-supported spermine oligomers
References and notes
1. Voirin, E.; Behr, J.-P.; Kotera, M. Nat. Protocols 2007, 2, 1360.
2. Pons, B.; Kotera, M.; Zuber, G.; Behr, J.-P. ChemBioChem 2006, 7, 1173.
3. Nothisen, M.; Kotera, M.; Voirin, E.; Remy, J.-S.; Behr, J.-P. J. Am. Chem. Soc.
2009, 131, 17730.
4. Noir, R.; Kotera, M.; Pons, B.; Remy, J.-S.; Behr, J.-P. J. Am. Chem. Soc. 2008, 130,
13500.
5. Moreau, V.; Voirin, E.; Paris, C.; Kotera, M.; Nothisen, M.; Remy, J.-S.; Behr, J.-P.;
Erbacher, P.; Lenne-Samuel, N. Nucleic Acids Res. 2009, 37. e130/1–e130/14.
6. Dempcy, R. O.; Browne, K. A.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
6097.
[7]. Park, M.; Bruice, T. C. Bioorg. Med. Chem. Lett. 2010, 20, 3982. and references
cited therein.
8. Schneider, S. E.; Bishop, P. A.; Salazar, M. A.; Bishop, O. A.; Anslyn, E. V.
Tetrahedron 1998, 54, 15063.
9. Manimala, J. C.; Anslyn, E. V. Eur. J. Org. Chem. 2002, 3909. and references cited
therein.
10. Zhang, Z.; Pickens, J. C.; Hol, W. G. J.; Fan, E. Org. Lett. 2004, 6, 1377.
11. Deglane, G.; Abes, S.; Michel, T.; Prévot, P.; Vives, E.; Debart, F.; Barvik, I.;
Lebleu, B.; Vasseur, J.-J. ChemBioChem 2006, 7, 684.
12. Pak, J. K.; Hesse, M. Helv. Chim. Acta 1998, 81, 2300.
13. Lang, H.; Gottlieb, M.; Schwarz, M.; Farkas, S.; Schulz, B. S.; Himmelsbach, F.;
Charubala, R.; Pfleiderer, W. Helv. Chim. Acta 1999, 82, 2172.
14. Yield of single step are usually from 30% to 37%.
Oligonucleotide syntheses were performed as follows: solid-sup-
ported spermine oligomers were detritylated before being loaded
onto the synthesizer. Junction between spermine oligomer and oli-
gonucleotide was achieved by creating phosphoramidate function-
ality. To this end, the first nucleotide was coupled with increased
reaction time (cycle X). To carry out a decamer synthesis, standard
instrumental protocol for a 1 lmol DNA synthesis was therefore im-
proved by: (1) increasing wash volume after TCA deblocking (total
volume ꢃ 4 mL) for complete DMT recovery, (2) extending capping
cycle time (3 min), (3) modifying cycle X to draw phosphoramidite
from position C instead of position 5. All oligonucleotides were syn-
thesized with final DMT-ON condition by programming the follow-
ing sequence: 30-XACCGTAGCG-DMT-50. Average coupling yields
were evaluated by measuring the 503 nm absorbance of recovered
DMT fractions diluted to 100 mL by 3% TCA/DCM solution. These
yields were comprised between 96.1% and 99.3%.
4.5. Cleavage from the support and purification
15. In a preliminary study, it was shown that the solid phase guanidine link
forming reaction on 50-aminolinker conjugated oligonucleotides can be done
for the first coupling while further coupling is not efficient presumably because
of partial deprotection of phosphate group during base catalyzed coupling
reaction.
The resin was then transferred to a screwcap tube and oligonu-
cleotide–oligospermine was cleaved from the support by action of
0.5 mL of concentrated ammonia (28% in water) for 24 h at rt. The
solution was next lyophilized and Npeoc protecting groups were
removed from the guanidino-spermine tail by suspending oligonu-
cleotide conjugates in a solution of DBU 1 M in acetonitrile
(0.2 mL) at 55 °C overnight and 48 h at room temperature. Acetoni-
trile from the resulting products was removed by lyophilisation
and the samples were dissolved in H2O mQ (0.4 mL). DMT-oligonu-
cleotide–oligospermines were then purified on a PolyPak II reverse
phase cartridge. Standard procedure4 was followed for the purifica-
tion of those conjugates, as previously described, except for the
elution step, which was accomplished by successive elutions of
20% acetonitrile in water (1 mL) and ammonia (1/20 or 1/10;
2 ꢁ 1 mL). Oligonucleotide-containing fractions were pooled. Addi-
tional desalting step was proven necessary because of residual DBU
and was performed on a NAP-5 column, according to the manufac-
turer’s protocol.
16. Coupling yields ranged between 70% and 88%.
17. Phosphoramidate links were formed. Phosphoramidate functions are known to
be resistant to basic conditions so that the oligonucleotide conjugate could be
cleaved and deprotected under standard conditions. See: Letsinger, R. L.;
Mungall, W. S. J. Org. Chem. 1970, 35, 3800.
18. We preferred two-step procedure. We observed that on-column Npeoc
deprotection with DBU/ACN induces a significant amount of ODN cleavage
from CPG.19–21
19. Stengele, K.-P.; Pfleiderer, W. Tetrahedron Lett. 1990, 31, 2549.
20. Browne, T.; Pritchard, C. E.; Turner, G.; Salisbury, S. A. J. Chem. Soc., Chem.
Commmun. 1989, 891.
21. Ferrer, E.; Neubauer, G.; Mann, M.; Eritja, R. J. Chem. Soc., Perkin Trans. 1 1997,
2051.
22. Damha, M. J.; Ogilvie, K. K. In Protocols for Oligonucleotides and Analogs.
Synthesis and Properties; Agrawal, S., Ed.; Humana Press Inc.: Totowa, New
Jersey, 1993; p 81. Chapter 5.